It was reported that Notch signaling is an evolutionarily highly conserved in vertebrate and invertebrate animals and plays a very pivotal role in determining the fate of cells in the initial stage of development. Notch signaling is known as a major factor that regulates the differentiation of neural cells, intraocular cells, lymphocytes, muscular cells, hematocytes and the like and is also involved in the development of blood vessels. Mammals have four Notch receptors (Notch 1, 2, 3 and 4), and each of Notch receptors is synthesized as a protein having a size of 300-350 kDa and cleaved at the S1 site by furin-like convertase in the Golgi to form a heterodimer on the cell surface. In addition, four Notch ligands (jagged-1/2 and delta-like ligand (DLL) 1/3/4) were found in mammals.
The Notch receptors and ligands are all membrane proteins and bind at the interface between two adjacent cells to induce Notch signaling. When cells come into contact with each other, the extracellular domains come into direct contact with each other to induce signaling, and cell responses that differ depending on combinations of ligands and receptors appear. When ligands and Notch receptors bind to each other in Notch signaling, the Notch receptors are structurally changed, and then undergo two sequential proteolytic cleavages. The first proteolytic cleavage begins with the cleavage of the extracellular domain (S2 site) by the metalloprotease ADAM10/17 (a disintegrin and metalloprotease 10/17)/TACE (TNF-α converting enzyme). When the S2 site is cleaved, the S3 site of transmembrane domain of the Notch receptor is then cleaved. The second proteolytic cleavage is mediated by a γ-secretase complex having five subunits. The γ-secretase complex is composed of presenilin 1, presenilin 2, nicastrin, Pen-2, and Aph1. After the two proteolytic cleavages, the Notch intracellular domain (NICD) is released and migrates into the nucleus. In the nucleus, the NICD binds to the transcriptional suppressor CSL (CBF-1/Suppressor of Hairless/Lag-1) to replace the corepressor (CoR) that has been bound to the CSL. The NICD/CSL complex recruits the co-activator (CoA) MAML (mastermind-like) or p300 to activate and induce the expression of Notch target genes such as cyclin D1, p21, NF-κB, c-Myc, pre-Ta (pre-T cell receptor alpha chain), GATA3, NRARP and Deltex1.
Activated Notch signaling is known to induce tumorigenesis in various tumor models. When the activated Notch NICD was expressed in rat hematopoietic cells, T-cell leukemia/lymphomas occurred and about 50% of activated Notch 1 was found in about 50% of T-ALL (T-cell acute lymphoblastic leukemia) (Weng A P et al., Science 2004; 306:71-269). In addition, in the case of breast cancer, Notch 4 receptor was found to be overexpressed in rats (Czech II) introduced with MMTV (mouse mammary tumor virus), and the occurrence of a mammary gland tumor in these rats was reported (Gallahan D et al., Journal of Virology 1987; 61:66-74). It was reported that Notch receptors and ligands and Notch signaling targets are activated in various cancers such as cervical cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer and prostate cancer (Miele L et al., Clin Cancer Research 2006; 12(4):1074-79), and it is known that Notch 1 receptor is associated with worse prognosis on breast cancer patients (Reedijk M et al., Cancer Research 2005; 65:8530-7) and associated with the metastasis of prostate cancer (Santagata S et al., Cancer Research 2004; 64:6854-7).
Delta-like 4 (D14) or delta-like ligand 4 (DLL4) (hereinafter referred to as “DLL4”) is one of delta-class ligands that bind to Notch proteins which are overexpressed in vascular endothelial cells. It is known as a major factor that regulates angiogenesis. DLL4 particularly binds to Notch 1 or Notch 4 receptor which is overexpressed in vascular endothelial cells. It is known that DLL4 is highly overexpressed in cancer blood vessels, although it is also expressed in normal blood vessels (Reinacher-Schick A et al., Nat Clin Pract Gastroenterol Hepatol 2008; 5(5):250-67). Angiogenesis refers to the mechanism by which new blood vessels are formed from the pre-existing blood vessels. Particularly, in tumors, angiogenesis is caused by angiogenic factors such as VEGF in order to supply oxygen and nutrients to the hypoxia area of cancer tissue. It is known that angiogenesis in tumors plays an important role not only in the growth of the tumor, but also in the metastasis of the tumor. When Notch signaling by DLL4 in tumors is blocked, angiogenesis cannot be easily controlled, and thus the growth of the tumors can be inhibited. In addition, when Notch signaling by DLL4 is inhibited, autoimmune disease can be treated by increasing the number of regulatory T cells (Treg) (US Patent Publication No. 2011-0189200). For these reasons, DLL4 becomes a new target in the treatment of cancers and autoimmune diseases.
In order to treat cancer or autoimmune disease by targeting DLL4, studies on the inhibition of Notch signaling in various portions have been conducted. Examples thereof include the receptor decoy that interferes with Notch/ligand bindings, a γ-secretase inhibitor that is involved in the cleavage of Notch proteins in Notch signaling, and miRNA or siRNA for inhibiting either proteins involved in Notch signaling or Notch target genes. Among these various methods for inhibiting Notch signaling, studies on monoclonal antibodies that bind Notch ligands capable of acting in the initial stage of Notch signaling have been of increasing importance. Particularly, for clinical studies, there has been a demand for the development of a human monoclonal antibody which can bind specifically to human DLL4 and can effectively inhibit DLL4/Notch receptor interactions while minimizing the risk of immunogenicity.